Development of a multiplexed assay in kinetoplastid parasites to identify probes for glycolysis

开发动质体寄生虫多重测定法来鉴定糖酵解探针

• 批准号: 10774367
• 负责人: KENNETH A CHRISTENSEN
• 金额: $ 6.61万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-03 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10774367
• 关键词: Address; Adopted; Antiparasitic Agents; Area; Biological Assay; Biology; Biosensor; Cells; Cellular Assay; Chemicals; Chemistry; Collection; Development; Disease; Eukaryota; Evaluation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Fructose; Future; Gluconeogenesis; Glucose; Glycolysis; Glycosome; Goals; Health; Human; In Vitro; Individual; Lead; Leishmania; Leishmania donovani; Libraries; Link; Maps; Measures; Metabolic; Metabolic Pathway; Metabolism; Methodology; Methods; Molecular Genetics; Monitor; Nature; Organism; Outcome; Outcomes Research; Parasites; Parasitic infection; Pathway interactions; Permeability; Phenotype; Proteins; Proteomics; Pyruvate; Recombinants; Reporter; Role; Series; Solubility; Stage Grouping; Time; Trypanosoma brucei brucei; Validation; Visualization; Work; World Health Organization; carbohydrate metabolism; cell type; cellular targeting; chemical property; cytotoxicity; genetic approach; glucose metabolism; glucose uptake; high throughput screening; improved; inhibitor; innovation; insight; member; multiplex assay; neglected tropical diseases; novel; novel strategies; pathogen; reverse genetics; screening; sensor; small molecule; tool

Project Summary/Abstract Parasite infection with members of the class Kinetoplastea, including Trypanosoma brucei and Leishmania spp., places a tremendous burden on human health, with an estimated ~1.4 million cases of disease recorded in 2015 (World Health Organization). Despite the widespread impact of these organisms, there remain major gaps in our understanding of fundamental parasite biology. For example, it is appreciated that glucose is a critical metabolite that can also serve to regulate important developmental pathways in the parasites. However, our understanding of these areas, particularly in the context of living parasites, is extremely limited. We propose to elucidate key mechanistic aspects of kinetoplastid glucose metabolism in kinetoplastids using a validated, yet unprecedented, screening assay platform based on live parasites expressing protein sensors that measure glucose uptake, distribution, and metabolism. We will adopt these cells to a novel and effective multiplexed high-throughput screening assay to identify small molecule probes that disrupt glucose uptake, distribution and metabolism in live parasites. This unique approach will simultaneously identify inhibitors of glycolysis that possess chemical properties required for delivery to the cellular target. Hits from the screening of a structurally diverse 100,000 compound collection will be validated by resynthesis, reconfirmation and counter- screening. Target identification, which will be facilitated by mapping the impact of the inhibitor on the pathway using the read-out from the assay, will be confirmed using reverse genetic and proteomic approaches. Last, the identified small molecule glycolytic probe inhibitors will be improved by SAR and their activity against parasites scored. Expected outcomes include development of a screening strategy that will be applicable to many types of cells, along with the identification of validated probes that dissect an essential parasite metabolic pathway.

项目摘要/摘要 包括布氏锥虫和利什曼原虫在内的动门螺纲成员的寄生虫感染 给人类健康带来巨大负担，2015年估计有约140万病例记录在案 (世界卫生组织)。尽管这些生物具有广泛的影响，但在我们的 对基础寄生虫生物学的理解。例如，人们认识到葡萄糖是一种关键的代谢物 这也可以用来调节寄生虫的重要发育途径。然而，我们对此的理解 这些地区，特别是在生活寄生虫的背景下，极其有限。 我们建议用一种新的方法来阐明动质体内葡萄糖代谢的关键机制。 经过验证但史无前例的基于表达蛋白质传感器的活体寄生虫的筛选分析平台 测量葡萄糖的摄取、分布和代谢。我们将以一种新颖和有效的方式采用这些细胞 多重高通量筛选试验以确定干扰葡萄糖摄取的小分子探针， 活体寄生虫的分布和代谢。这一独特的方法将同时识别出 糖酵解具有传递到细胞靶标所需的化学性质。放映一部电影的热门歌曲 将通过重新合成、重新确认和反合成来验证结构多样化的100,000个化合物集合 放映。靶标识别，这将通过绘制抑制物对途径的影响图来促进 使用从检测中读出的数据，将使用反向遗传学和蛋白质组学方法进行确认。最后， 识别的小分子糖酵解探针抑制剂将通过SAR及其抗寄生虫活性得到改善 得分了。预期成果包括制定适用于多种类型的筛查战略 细胞，以及对剖析寄生虫重要代谢途径的有效探针的鉴定。